This invention relates to nucleic acid sequences and proteins involved in hypohidrotic ectodermal dysplasia (HED) and hair follicle induction.
Hereditary ectodermal dysplasia is an inherited disorder that affects the development of ectodermally derived structures, such as the hair, teeth and sweat glands. The hidrotic form of the disease is characterized by poorly developed teeth and hair. The anhidrotic or hypohidrotic form of the disease further affects the development of sweat glands, which interferes with the ability to sweat, and the maintenance of thermoregulatory homeostasis. Both X-linked and autosomal dominant and recessive forms of the disease have been described.
X-linked hypohidrotic ectodermal dysplasia (XLHED; McKusick""s number 305100), the most common form of the ectodermal dysplasias, results in the abnormal development of teeth, hair and eccrine sweat glands. Identification of the gene that is defective in this disease would help explain the molecular basis of XLHED, as well as the molecular mechanisms involved in normal tooth, hair and eccrine sweat gland development. Identification of the gene would also permit mutation testing for XLHED in potentially affected males and carrier females.
Heterozygote carriers of XLHED may have minor or moderate degrees of hypodontia, hypotrichosis and hypohidrosis, although many show no obvious clinical manifestations. This clinical variation, presumably caused by random X-inactivation (Lyonization), makes accurate diagnosis of carrier females difficult. Although indirect testing for carrier status is possible by linkage analysis in informative families (Zonana 1993), carrier detection by this method is impossible in families with single affected individuals, male or female, whose disorder may be the result of a de novo mutation. Detection of mutations within the EDA1 gene would also be advantageous in families with only a single affected sibship, because a rarer autosomal recessive form of the disorder (ARHED) is clinically indistinguishable from XLHED in affected males (Munoz et al. 1997).
A gene identified as EDA1 has been isolated by positional cloning (Kere et al. 1996). A single 858 bp cDNA, representing a full length transcript composed of 2 exons, was identified from an adult sweat gland cDNA library. In situ analysis showed that the EDA1 gene was expressed in hair follicles and the epidermis of adult skin. The putative gene product is a 135 amino acid protein, which has no clear homology to other proteins (see U.S. Pat. No. 5,700,926). The protein is predicted to contain a single transmembrane domain, and fractionation studies of transfected cell lines showed that the protein product is localized to the plasma membrane (Ezer et al. 1997). Yeast artificial chromosomes (YACs) containing at least a portion of the human EDA1 gene were disclosed in U.S. Pat. No. 5,556,786.
A syndrome similar to HED, with anhidrosis and absence of sweat glands, is known in the mouse, in which the mutant gene is called Tabby (Ta). Consistent with the map position in humans, the Ta gene has been mapped in the syntenically corresponding region in the X chromosome of the mouse (Brockdorff et al. 1991). Other murine forms of the disease include those found in the downless (dl) mutants, in which the disorder is not X-linked.
The present invention has been made possible by the discovery that there are previously unidentified alternative transcripts of the EDA1 gene. The inventors recognized that while the full length of the EDA1-I cDNA was 858 bp, a predominant 5-6 kb transcript was detected in several human tissues by Northern analysis. Mutation screening of 173 unrelated families with XLHED showed that only 7% of the families had likely mutations within exon 1, and none had variants within exon 2. Moreover, cDNAs from the homologous murine gene, Tabby (Ta), were found to include alternative exons.
The present invention includes an EDA1 cDNA splice-form (Seq. I.D. No. 1) that is homologous to the Ta cDNA (Seq. I.D. No. 3), and codes for a second isoform of the EDA1 protein (isoform II or EDA1-II). Nearly all of the mutations associated with XLHED are located within the exons identified in this new splice-form. These results show that EDA1 isoform II is essential for hair, tooth and eccrine sweat gland morphogenesis. In addition, the identification of the additional exons permits direct molecular diagnostic testing for XLHED by mutation analysis.
Also disclosed is the nucleic acid sequence encoding the human gene EDA1-II sequence (Seq. ID. NOS. 5-11), which is predicted to encode a 391 amino acid protein (Seq. I.D. No. 2). The EDA1-II gene is predicted to encode a protein that is related to the TNF family of proteins, and acts as either a membrane-associated or soluble ligand. The biologically active domains of the EDA1-II protein are within approximately the C-terminal 240 amino acids (and particularly residues 133-391, and especially residues 239-391 of EDA1-II) and includes a Furin recognition sequence, a Gly-X-Y repeat, and a region of structural homology to TNF proteins. This predicted active region is unique to this isoform, with none of the amino acids included within the previously described EDA1 sequence. The DNA sequence that codes for this isoform (Seq. I.D. No. 1) is also unique.
The EDA1-II protein is an essential component of a signaling pathway that is required for the normal development of hair follicles, teeth, sweat glands and mammary glands. A form of the protein is present in humans, mice, cow and dog, and is likely to serve the same or nearly identical roles in each of these organisms. The invention includes the use of the human EDA1-II gene sequence, as it applies to the use of commercial and clinical diagnostic testing for ectodermal dysplasia (ED). The invention also includes use of an EDA1-II gene sequence for the production of EDA1-II protein, as a therapeutic substance stimulating the growth of hair, teeth, skin, and sweat glands. Potential medical or cosmetic benefits of EDA1-II include the stimulation of hair growth, including cases of alopecia (balding) or skin grafts. As such, EDA1-II may be applied as a purified protein or nucleic acid (DNA or RNA), delivered as topical substance, or injected into the skin, alone or in combination with a pharmaceutical carrier.
The EDA1-II isoform (or active subsequences thereof, particularly the C terminal 240 amino acid residues, or residues 133-391, particularly 239-391), may also be used as a stimulant for tooth growth, either in cases of tooth loss or of natural absence of teeth. The protein may be used to stimulate tooth growth in humans directly, or alternatively in tissue culture (artificial) conditions, with subsequent introduction of teeth into humans or other organisms. The EDA1-II protein or EDA1-II gene may also be useful for the stimulation of eccrine sweat gland development, for example in individuals for whom the normal sweating mechanism is compromised by disease or surgery.
The protein may also be used to stimulate the growth of mammary epithelial tissue, either for reconstructive or cosmetic purposes. Alternatively, methods that block the production of the protein, for example antisense or antibody approaches, may be useful for inhibiting breast epithelial cell proliferation. Blocking EDA1-II activity may provide an effective therapeutic approach to slow or to inhibit the spread of breast cancer malignancies. The EDA1-II gene and gene product itself may also be useful in promoting or maintaining differentiation of breast epithelium.
The present invention also provides an isolated human nucleic acid molecule which may be able to correct the cellular defect characteristic of HED, including XLHED. It is shown that XLHED patients carry mutations in the genomic copies of this nucleic acid molecule. Orthologs of the disclosed nucleic acid molecule from other species are also provided, which may be able to correct the effects of the mutation. Such homologous proteins may have 95% or 98% identity to the EDA1-II isoform. Also included are DNA sequences that code for EDA1-II, including probes and primers for these DNA molecules.
More specifically, the invention provides an isolated human cDNA, herein referred to as the human ectodermal dysplasia gene, isoform II sequence (EDA1-II) cDNA for correcting or improving abnormal biochemical activities of cells in which it is expressed, for example in human cells derived from XLHED patients. Also provided by this invention is the amino acid sequence of the protein ligand encoded by this cDNA, and DNA and amino acid sequences of receptors (referred to as murine downless, dl, and human DL) for the ligands to bind as agonists. Murine and human cDNA and amino acid sequences for the dl (murine) and DL (human) receptors are provided in Seq. I.D. NOS. 12, 19, 18 and 17.
The invention also provides an isolated human nucleic acid molecule which may be able to correct the cellular defect characteristic of HED, including non-X-linked, autosomal forms of the disease. More specifically, the invention provides an isolated cDNA, referred to as the DL gene, for correcting or improving biochemical activities in human cells derived from ectodermal dysplasia patients in which it is expressed. Also provided are the amino acid sequences of the proteins encoded by these cDNAs.
Having provided the nucleotide sequence of the human EDA1-II cDNA and the dl and DL receptors, correspondingly provided are the complementary DNA strands of these cDNA molecules and DNA molecules which hybridize under stringent conditions to these cDNA molecules, or their complementary strands. Such hybridizing molecules include DNA molecules differing only by minor sequence changes, including nucleotide substitutions, deletions and additions. Guidance about making such mutations, while maintaining biological activity of the proteins, is provided by the illustration of mutations that interfere with biological function. Further guidance is provided by comparison of the sequences from different species, which illustrate highly conserved sequences. Moreover, the disclosure of the dl and DL receptors permits the construction of a particularly convenient assay for determining whether a variant ligand binds to the receptor, and would be a candidate agonist or antagonist.
Through the manipulation of the disclosed nucleotide and amino acid sequences by standard molecular biology techniques, variants of the EDA1-II agonist, and the dl and DL receptors, may be made which differ in precise amino acid sequence from the disclosed proteins, yet which maintain the basic functional characteristics of the disclosed proteins, or which are selected to differ in some characteristics from these proteins. Variants can also be made which interfere with receptor action, and therefore act as antagonists.
Also comprehended by this invention are isolated oligonucleotides comprising at least a segment of the disclosed cDNA molecules or the complementary strands of these molecules, such as oligonucleotides which may be employed as DNA hybridization probes or DNA primers useful in the polymerase chain reaction. Hybridizing DNA molecules and variants of the EDA1-II cDNA and the dl and DL cDNAs may readily be created by standard molecular biology techniques. Such oligonucleotides may be used for detecting an enhanced susceptibility of an individual to ectodermal dysplasia. Specifically, the oligonucleotides of the present invention may be used for molecular diagnostic testing for XLHED and HED by mutation analysis.
Having provided the isolated human EDA1-II ligand sequence, and the mammalian dl and DL receptor sequences, this invention also includes genomic sequences from which these cDNAs are derived. The exon sequences of EDA1-II are shown in Seq. I.D. NOS. 5-11, and the exon/intron sequences of DL are shown in Table 5 (Seq. I.D. NOS. 94-116). Also provided by the present invention are recombinant DNA vectors comprising the disclosed DNA molecules, and transgenic host cells containing such recombinant vectors.
The present invention also provides methods for using the disclosed cDNAs, the corresponding genomic sequence, and derivatives thereof, and of the expressed protein, and derivatives thereof, in aspects of diagnosis of ectodermal dysplasia, and detection of carriers. One particular embodiment of the present invention is a method for screening a subject to determine if the individual carries a mutant EDA1-II gene or a mutant dl or DL gene. The method includes detecting the presence of nucleotide differences between the sequence of the individual""s EDA1-II, dl or DL gene ORF compared to the EDA1-II, dl or DL cDNA or genomic sequence disclosed herein, or the presence of nucleotide differences between the individual""s dl or DL gene ORF compared to the dl or DL cDNA sequence disclosed herein, and determining whether any such sequence differences will result in the expression of an aberrant gene product in the individual. The step of detecting nucleic acid sequence differences may be performed using several techniques including: hybridization with oligonucleotides (including, for example, the use of high-density oligonucleotide arrays); PCR amplification of the gene or a part thereof using oligonucleotide primers; RT-PCR amplification of the EDA1-II, dl or DL RNA, or a part thereof using oligonucleotide primers, and direct sequencing of the EDA1-II, dl or DL gene of the individual""s genome using oligonucleotide primers.
In a specific embodiment, the oligonucleotides used for detecting a mutation in the EDA1-II or DL gene of an individual are selected from the group consisting of SEQ. I.D. NOS 20-29, SEQ. I.D. NOS 74-93 and SEQ. I.D. NOS 117-118. XLHED. In another embodiment, methods for detecting a mutation in an individual suffering from XLHED or an autosomal form of ectodermal dysplasia, are provided. Such methods involve incubating at least one of the oligonucleotides listed above with a nucleic acid preparation of the individual under conditions such that the oligonucleotide primer specifically hybridizes to an EDA1-II gene or a DL gene present in the nucleic acid preparation to form an oligonucleotide:EDA1-II gene or DL gene complex. This complex is then used to amplify the nucleic acid of the EDA1-II or DL gene defined by the oligonucleotide primer used. After this amplification, the presence or absence of mutations within the EDA1-II or DL gene complex are detected using any method known to those skilled in the art, such as SSCP, ASO, direct sequencing or dideoxyfingerprinting analysis. The presence of a mutation(s) indicates an enhanced susceptibility of the individual to ectodermal dysplasia.
The disclosed sequences have also been useful in the creation and study of mutations in the EDA1-II, dl or DL locus that affect biochemical activity of the protein, which in turn has yielded valuable information about the biochemical pathways underlying the disease, as well as information about epithelial mesenchymal signaling.
A further aspect to the present invention is a preparation comprising specific binding agents, such as antibodies, that specifically detect the EDA1-II, dl or DL proteins. Such specific binding agents may be used in methods for screening a subject to assay for the presence of a mutant EDA1-II, or DL gene. One exemplary method comprises providing a biological sample of the subject which sample contains cellular proteins, and providing an immunoassay for quantitating the level of EDA1-II, dl or DL protein in the biological sample.
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.